Design and synthesis of new ruthenium polypyridyl complexes with potent antitumor activity in vitro

Article abstract of DOI:10.1016/j.saa.2019.05.037

We herein report the synthesis, characterization and anticancer activity of BTPIP (2-(4-(benzo[b]thiophen-2-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its four ruthenium(II) polypyridyl complexes [Ru(N–N)₂(BTPIP)](ClO₄)

Full text of DOI:10.1016/j.saa.2019.05.037

Accepted Manuscript
Design and synthesis of new ruthenium polypyridyl complexes
with potent antitumor activity in vitro
Guang-Bin Jiang, Wen-Yao Zhang, Miao He, Yi-Ying Gu, Lan
Bai, Yang-Jie Wang, Qiao-Yan Yi, Fan Du
PII:
S1386-1425(19)30513-X
https://doi.org/10.1016/j.saa.2019.05.037
SAA 17132
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 17 March 2019
Revised date:
Accepted
date:
6 May 2019
1
2 May 2019
Please cite this article as: G.-B. Jiang, W.-Y. Zhang, M. He, et al., Design and synthesis
of new ruthenium polypyridyl complexes with potent antitumor activity in vitro,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/
1
0.1016/j.saa.2019.05.037
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ACCEPTED MANUSCRIPT
Submitted to Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Design and Synthesis of New Ruthenium Polypyridyl Complexes with
Potent Antitumor Activity in Vitro
a,*
b
b
b
b
Guang-Bin Jiang,
Wen-Yao Zhang, Miao He, Yi-Ying Gu, Lan Bai,
b
b
b
Yang-Jie Wang, Qiao-Yan Yi, Fan Du
a
Guangxi Key Laboratory of Electrochemical and Magneto-chemical Function
Materia, College of Chemistry and Bioengineering, Guilin University of Technology,
Guilin, 541004, China.
b
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006,
PR China
*
Corresponding authors. Tel: +86-773-899-1304; fax: +86-773-899-1304.
E-mail address: jianggb@glut.edu.cn (G.B. Jiang).
1

ACCEPTED MANUSCRIPT
Abstract We herein report the synthesis, characterization and anticancer activity of
BTPIP (2-(4-(benzo[b]thiophen-2-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline)
and its four ruthenium(II) polypyridyl complexes [Ru(N-N) (BTPIP)](ClO ) (N-N =
2
4 2
bpy = 2,2'-bipyridine, Ru(II)-1; phen = 1,10-phenanthroline, Ru(II)-2; dmb =
,4′-dimethyl-2,2′-bipyridine, Ru(II)-3; dmp = 2,9-dimethyl-1,10-phenanthroline,
4
Ru(II)-4). The DNA binding behaviors reveal that the complexes bind to calf thymus
DNA by intercalation. Cytotoxicity of the complexes against A549, HepG-2,
SGC-7901 and Hela cells were evaluated in vitro. Complexes Ru(II)-1, Ru(II)-2,
Ru(II)-3, Ru(II)-4 show moderate activity on the cell proliferation in A549 cells with
IC values of 9.3 ± 1.2, 12.1 ± 1.6, 10.3 ± 1.6, 8.9 ± 1.2 μM, respectively. Apoptosis
5
0
assessment, intracellular mitochondrial membrane potential (MMP), location in
mitochondria, reactive oxygen species (ROS), cell invasion assay and cell cycle arrest
were also performed to explore the mechanism of this action. When the concentration
of the ruthenium(II) complexes is increased, the amount of reactive oxygen species
increases obviously and the mitochondrial membrane potential decreases dramatically
in A549 cells. Most importantly, the ruthenium(II) polypyridyl complexes could arrive
the cytoplasm through the cell membrane and accumulate in the mitochondria. These
results showed that the ruthenium(II) complexes could induce apoptosis in A549 cells
through an ROS-mediated mitochondrial dysfunction pathway.
Keywords: Ruthenium(II) complex; antitumor; intercalation; reactive oxygen species;
mitochondrial membrane potential.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Platinum-based anticancer drugs, including cisplatin, carboplatin and oxaliplatin,
have been widely used in the field of antitumor chemotherapies and continue to
represent the mainstay of therapy for various cancer types [1-8]. However,
platinum-based anticancer drugs also show many shortcomings, such as
nephrotoxicity, myelotoxicity, neural damage, ototoxicity, and strong resistance,
which prompted to search the alternative metal-based anticancer drugs with high
efficient and selectivity [9-13].
It was believed that the complexes containing ruthenium display low toxicity,
easily absorbed and rapidly excreted by the body, and will become one of the most
promising antitumor drugs [14-19]. Therefore, during the past decades, remarkable
efforts have been devoted to synthesis ruthenium-based anticancer drugs [20-28]. For
instance,
Wong
and
co-workers
(N-N
demonstrated
that
[
Ru(N-N) -(p-MOPIP)](PF ) ·2H O
=
2,2′-bipyridine
(bpy);
2
6 2
2
1
2
,10-phenanthroline (phen); 4,4′-dimethyl-2,2′-bipyridine (dmb); PIP
=
-phenylimidazo[4,5-f][1,10]phenanthroline) can induce mitochondria-mediated and
caspase-dependent apoptosis in various cancer cells [29]. The Maysinger group
reported that ruthenium(II)-letrozole complexes can effectively inhibit the
proliferation of MCF-7 and U251N cells [30]. In 2015, Chao found that four
ruthenium(II) complexes can utilize as mitochondria-targeted two-photon
photodynamic anticancer agents [31]. Very recently, the group of Liu described that
[
Ru(phen) (PFPIP)](ClO ) ], a Ru(II) complex with high antitumor activity, was able
2 4 2
3

ACCEPTED MANUSCRIPT
to effectively inhibit growth of HepG-2 cells [32].
It is well known that heterocyclic compounds have been reported to possess
remarkable anticancer, anti-inflammatory, antibacterial, antioxidant, antidiabetic,
immuomodulatory,
and
anti-HIV
activities
[23-35].
Among
them,
thiophene-containing compounds have been attracted more attention owing to their
wide range of biological properties [36-38]. Nonetheless, thiophene-containing
ruthenium complexes are rarely investigated as anticancer drugs. In continuation of
our interest in the field of antitumor, herein, a thiophene-containing ligand BTPIP and
its four ruthenium(II) polypyridyl complexes [Ru(bpy) (BTPIP)](ClO ) (Ru(II)-1),
2
4 2
[
[
Ru(phen) (BTPIP)](ClO ) (Ru(II)-2), [Ru(dmb) (BTPIP)](ClO ) (Ru(II)-3), and
2 4 2 2 4 2
Ru(dmp) (ETPIP)](ClO )
(Ru(II)-4) (Scheme 1) were synthesized and
2
4 2
1
13
characterized by H NMR, C NMR, HRMS and IR. The interaction of the Ru(II)
polypyridyl complexes with CT DNA was investigated via electronic absorption
titration, viscosity measurements and luminescence spectra. In addition, the anticancer
activities of these complexes were firstly examined in vitro against the A549, HepG-2,
SGC-7901 and Hela cancer cell lines. Most importantly, the possible antitumor
mechanism of the Ru(II) complexes were detected by determining the cells
morphological changes, cell cycle arrest, ROS levels and the decrease of the
mitochondrial membrane potentials.
2
. Experimental section
2
.1. Materials and methods
4

ACCEPTED MANUSCRIPT
All reagents and solvents were purchased commercially and used without further
purification unless otherwise noted. Calf thymus DNA (CT DNA) was obtained from
the Sino American Biotechnology Company. Ltd. Ultrapure MilliQ water was used in
all experiments. DMSO and RPMI 1640 were purchased from Sigma. Cell lines of
HeLa (Human cervical cancer cell line), SGC-7901 (human gastric carcinoma cells),
HepG-2 (Hepatocellular carcinoma cells) and A549 (Human lung carcinoma cells)
were purchased from the American Type Culture Collection. RuCl ·3H O was
3
2
obtained from the Kunming Institution of Precious Metals. 2,2'-Bipyridine,
,10-phenanthroline, 4,4′-dimethyl-2,2′-bipyridine, 2,9-dimethyl-1,10-phenanthroline
1
were obtained from the Guangzhou Chemical Reagent Factory.
Analytical thin layer chromatography was performed by using commercially
prepared 100-400 mesh silica gel plates (GF ) and visualization was effected at 254
2
54
nm. Mass spectra were recorded on a Thermo Scientific ISQ gas chromatograph-mass
spectrometer. The data of HRMS was carried out on a high-resolution mass
spectrometer (LCMS-IT-TOF). IR spectra were obtained either as potassium bromide
pellets or as liquid films between two potassium bromide pellets with a Bruker
1
TENSOR 27 spectrometer. H NMR spectra were recorded on a Varian-500
spectrometer with DMSO-d as solvent and tetramethylsilane (TMS) as an internal
6
standard at 400 MHz at room temperature.
2
2
.2. Synthesis of ligand and complexes
.2.1. Synthesis of 4-(benzo[b]thiophen-2-yl)benzaldehyde
5

ACCEPTED MANUSCRIPT
To a resealable Schlenk tube or alternatively, a screw-cap pressure tube, were
added 4-bromobenzaldehyde (1.0 mmol, 1.0 equiv), Pd(OAc)2 (5 mol %),
benzo[b]thiophen-2-ylboronic acid (1.5 mmol, 1.5 equiv), K CO (2 mmol, 2 equiv),
2
3
toluene (6 mL) and a stir bar. The reaction vessel was fitted with a rubber septum, and
was evacuated and back-filled with nitrogen. The reaction tube was sealed and
immersed in a preheated oil bath at 100 °C for 12 h and the solution was stirred with
the aid of a magnetic stirrer. After attaining ambient temperature, the reaction mixture
was diluted with ethyl acetate and filtered through a plug of silica gel. The filtrate was
concentrated and the resulting residue was purified by column chromatography (silica
gel, petroleum ether/EtOAc) to give the desired aldehydes.
2
.2.2. Synthesis of ligand (BTPIP)
A mixture of 1,10-phenanthroline-5,6-dione (100.0 mg, 0.5 mmol),
-(benzo[b]thiophen-2-yl)benzaldehyde (119.0 mg, 0.5 mmol), ammonium acetate
4
(15 mmol, 1156.2 mg) and acetic acid (30 mL) was refluxed with stirring for 4 h. The
cooled solution was diluted with water and neutralized with concentrated aqueous
ammonia. The brown precipitate was collected and purified by column
chromatography on silica gel (60~100 mesh) with ethanol as eluent to give the
compound as a brown yellow powder. Yield: 188.3 mg, 88%. Anal. Calc for
C H N S: C, 75.68; H, 3.76; N, 13.07%. Found: C, 75.80; H, 3.71; N, 12.99%; IR: ν
27
16
4
=
3101, 2102, 1916, 1793, 1691, 1597, 1588, 1480, 1358, 1312, 1188, 1121, 953, 819,
-1 1
7
36, 682 cm ; H NMR (400 MHz, DMSO-d ) δ 9.03 (d, J = 4.0 Hz, 2H, a, a'), 8.94
6
6

ACCEPTED MANUSCRIPT
(d, J = 8.1 Hz, 2H, c, c'), 8.40 (dd, J = 22.3, 8.3 Hz, 2H, j, k), 7.99 (d, J = 10.1 Hz, 5H,
13
d, e,f, g, l), 7.88-7.80 (m, 3H, h, b, b'), 7.40 (t, J = 6.8 Hz, 1H, i); C NMR (100 MHz,
DMSO-d ) δ 192.8 (1C, n'), 172.5 (1C, p), 150.4 (1C, a), 148.1 (1C, a'), 143.7 (1C, u),
6
1
42.9 (1C, t), 142.1 (1C, r), 140.9 (1C, n), 139.2 (1C, x), 134.8 (1C, v), 130.8 (1C,
c), 130.4 (1C, c'), 130.2 (2C, d, g), 127.4 (1C, e, f), 127.0 (1C, s), 126.9 (1C, s'),
1
1
25.9 (1C, j), 125.5 (1C, k), 125.4 (1C, o), 124.7 (1C, i), 124.4 (1C, l), 123.8 (1C, o'),
23.0 (1C, b), 122.9 (1C, b'), 121.2 (1C, h); HRMS (ESI) m/z: calcd for C H N S
27
17
4
+
[
M+H] , 429.1168; found 429.1169.
2
.2.3. Synthesis of [Ru(bpy) (BTPIP)](ClO ) (Ru(II)-1)
2 4 2
A mixture of cis-[Ru(bpy) Cl ]·2H O (158.7 mg, 0.3 mmol) and BTPIP (128.4 mg,
2
2
2
o
0
.3 mmol) in ethylene glycol (12 mL) was heated at 150 C under argon for 8 h to
give a clear red solution. Upon cooling, a red precipitate was obtained by dropwise
addition of saturated aqueous NaClO solution. The crude product was purified by
4
column chromatography on neutral alumina with a mixture of CH CN-toluene (1:1,
3
v/v) as eluent. The red band was collected. The solvent was removed under reduced
pressure and a red powder was obtained. Yield: 259.2 mg, 83%. Anal. Calc for
C H Cl N O RuS: C, 54.24; H, 3.10; N, 10.77%. Found: C, 54.33; H, 3.02; N,
47
32
2
8
8
1
1
9
8
0.86%; UV-vis (5 μM; λmax, 467 nm); IR: ν = 3073, 1603, 1511, 1479, 1463, 1446,
-1
1
313, 1272, 953, 827, 808, 765, 726, 624 cm ; H NMR (400 MHz, DMSO-d ) δ
6
.05 (d, J = 8.1 Hz, 2H, a, a'), 8.81 (d, J = 8.2 Hz, 4H, 4, 4', 5, 5'), 8.43 (s, 4H, 1, 1', 8,
'), 8.36 (d, J = 7.9 Hz, 2H, c, c'), 8.19 (d, J = 5.0 Hz, 2H, d, g), 8.13 (d, J = 4.9 Hz,
7

ACCEPTED MANUSCRIPT
2
H, e, f), 8.04-7.89 (m, 7H, h, l, i, b, b', 3, 3'), 7.83-7.77 (m, 6H, k, j, 2, 2', 6, 6'), 7.40
1
3
(
dd, J = 14.7, 7.3 Hz, 2H, 7, 7'); C NMR (100 MHz, DMSO-d ) δ 158.8 (2C, n, n'),
6
1
8
1
1
1
58.5 (2C, 9, 9'), 153.3 (2C, 10, 10'), 153.2 (1C, p), 153.2 (2C, 1, 1'), 150.6 (2C, 8,
'), 147.8 (2C, a, a'), 147.7 (1C, u), 145.9 (1C, t), 142.8 (1C, r), 140.9 (2C, 3, 3'),
39.3 (2C, 6, 6'), 137.3 (1C, x), 135.3 (1C, v), 130.9 (2C, c, c'), 131.0 (2C, d, g),
30.9 (1C, e), 128.9 (1C, f), 127.9 (2C, s, s'), 127.6 (2C, 4, 4'), 127.1 (2C, 5, 5'),
26.9 (1C, j), 126.8 (1C, k), 126.6 (2C, o, o'), 126.4 (1C, i), 125.5 (1C, l), 125.5 (2C,
b, b'), 124.5 (2C, 2, 2'), 123.0 (2C, 7, 7'), 121.5 (1C, h); HRMS (ESI) m/z: calcd for
+
C H N SRu [M-2ClO -H] , 841.1442; found 841.1450.
47
31
8
4
2
.2.4. Synthesis of [Ru(phen) (BTPIP)](ClO ) (Ru(II)-2)
2 4 2
This complex was synthesized in an identical manner to that described for
complex Ru(II)-1, with cis-[Ru(phen) Cl ]·2H O [32,39] in place of
2
2
2
cis-[Ru(bpy) Cl ]·2H O. Yield: 254.8 mg, 78%. Anal. Calc for C H Cl N O RuS: C,
2
2
2
51 32
2
8
8
5
4
9
7
6.26; H, 2.96; N, 10.29%. Found: C, 56.41; H, 3.04; N, 10.21%; UV-vis (5 μM; λmax
,
65 nm); IR: ν = 3051, 1601, 1576, 1508, 1478, 1455, 1426, 1363, 1304, 1250, 1224,
-1 1
44, 843, 808, 750, 623, 528 cm ; H NMR (400 MHz, DMSO) δ 9.13 (dd, J = 24.7,
.9 Hz, 2H, a, a'), 8.90 (dd, J = 14.6, 8.1 Hz, 4H, 1, 1', 8, 8'), 8.47 (dd, J = 41.7, 7.2
Hz, 2H, 3, 3'), 8.24 (t, J = 7.8 Hz, 2H, 6, 6'), 8.13 (t, J = 7.7 Hz, 2H, c, c'), 8.00 (t, J =
9
7
.7 Hz, 4H, d, e, f, g), 7.96-7.81 (m, 7H, h, l, i, b, b', 4, 4'), 7.70-7.61 (m, 4H, 5, 5', 7,
1
3
'), 7.41 (d, J = 6.3 Hz, 4H, k, j, 2, 2'); C NMR (100 MHz, DMSO-d ) δ 157.3 (2C,
6
n, n'), 157.1 (2C, 9, 9'), 151.9 (2C, 10, 10'), 149.1 (1C, p), 149.2 (2C, a, a'), 149.3 (2C,
8

ACCEPTED MANUSCRIPT
8
1
1
1
1
, 8'), 145.2 (2C, a, a'), 145.1 (1C, u), 143.1 (1C, t), 141.0 (1C, r), 139.2 (1C, n),
38.4 (2C, 3, 3'), 138.2 (2C, 6, 6'), 134.5 (1C, x), 130.9 (1C, v), 128.4 (2C, c, c'),
28.2 (2C, d, g), 127.6 (2C, e, f), 127.7 (2C, 4, 4'), 126.8 (2C, 5, 5'), 126.2 (2C, s, s'),
26.3 (2C, 11, 11'), 125.4 (2C, 12, 12'), 125.5 (1C, j), 124.9 (1C, k), 125.0 (2C, o, o'),
24.8 (1C, i), 124.4 (1C, l), 123.0 (2C, 2, 2'), 121.0 (2C, 7, 7'), 115.4 (1C, h); HRMS
+
(
ESI) m/z: calcd for C H N SRu [M-2ClO -H] , 889.1443; found 889.1451.
5
1
31
8
4
2
.2.5. Synthesis of [Ru(dmb) (BTPIP)](ClO ) (Ru(II)-3)
2 4 2
This complex was synthesized in an identical manner to that described for
complex Ru(II)-1, with cis-[Ru(dmb) Cl ]·2H O [32,39] in place of
2
2
2
cis-[Ru(bpy) Cl ]·2H O. Yield: 266.6 mg, 81%. Anal. Calc for C H Cl N O RuS: C,
2
2
2
51 40
2
8
8
5
4
8
2
7
7
5.84; H, 3.68; N, 10.21%. Found: C, 55.73; H, 3.57; N, 10.28%; UV-vis (5 μM; λmax
,
76 nm); IR: ν = 3057, 2919, 1618, 1507, 1479, 1448, 1364, 1304, 1197, 1093, 953,
-1 1
25, 725, 566, 544 cm ; H NMR (400 MHz, DMSO) δ 9.06 (dd, J = 13.5, 8.2 Hz,
H, a, a'), 8.75 (d, J = 15.5 Hz, 4H, 3, 3', 4, 4'), 8.39 (dd, J = 22.9, 7.7 Hz, 2H, 1, 1'),
.98 (m, J = 31.6, 22.2, 5.8 Hz, 9H, 6, 6', c, c', d, e, f, g, h), 7.70 (s, 2H, l, i),
.48-7.36 (m, 6H, b, b', k, j, 2, 2'), 7.20 (s, 2H, 5, 5'), 2.58 (s, 6H, 7), 2.48 (s, 6H, 8);
1
3
C NMR (100 MHz, DMSO-d ) δ 156.8 (2C, n, n'), 156.7 (2C, 9, 9'), 153.6 (2C, 10,
6
1
1
1
0'), 151.0 (1C, p), 150.9 (2C, 1, 1'), 150.0 (2C, 6, 6'), 149.9 (2C, a, a'), 149.8 (1C, u),
45.6 (1C, t), 142.9 (1C, r), 140.9 (1C, n), 139.3 (2C, 11, 11'), 135.1 (2C, 12, 12'),
35.0 (1C, x), 130.7 (1C, v), 130.5 (2C, c, c'), 129.4 (2C, d, g), 129.0 (2C, e, f), 128.8
(2C, s, s'), 127.7 (2C, 3, 3'), 127.0 (2C, 4, 4'), 127.0 (1C, j), 126.5 (1C, k), 125.5 (2C,
9

ACCEPTED MANUSCRIPT
o, o'), 125.4 (1C, i), 124.4 (1C, l), 123.0 (2C, b, b'), 121.3 (2C, 2, 2'), 121.0 (2C, 5, 5'),
2
1.2 (2C, 7, 7'), 21.1 (2C, 8, 8'); HRMS (ESI) m/z: calcd for C H N SRu
51 39 8
+
[
M-2ClO -H] , 897.2069; found 897.2077.
4
2
.2.6. Synthesis of [Ru(dmp) (BTPIP)](ClO ) (Ru(II)-4)
2 4 2
This complex was synthesized in an identical manner to that described for
complex Ru(II)-1, with cis-[Ru(dmp) Cl ]·2H O [32,39] in place of
2
2
2
cis-[Ru(bpy) Cl ]·2H O. Yield: 237.1 mg, 69%. Anal. Calc for C H Cl N O RuS: C,
2
2
2
55 40
2
8
8
5
4
8
7.69; H, 3.52; N, 9.79%. Found: C, 57.56; H, 3.61; N, 9.72%; UV-vis (5 μM; λmax
,
79 nm); IR: ν = 3053, 1964, 1605, 1588, 1507, 1478, 1433, 1348, 1248, 1216, 1089,
-1 1
54, 809, 726, 622, 555 cm ; H NMR (400 MHz, DMSO) δ 8.93 (d, J = 8.3 Hz, 2H,
a, a'), 8.83 (d, J = 8.0 Hz, 2H, c, c'), 8.48-8.43 (m, 3H, 3, 3', l), 8.28 (t, J = 10.2 Hz,
3
2
H, 2, 2', i), 8.02-7.96 (m, 4H, 6, 6', 4, 4'), 7.94-7.87 (m, 3H, h, b, b'), 7.52-7.44 (m,
H, d, g), 7.43-7.34 (m, 6H, e, f, 5, 5', k, j), 7.19 (m, J = 34.5, 7.6 Hz, 2H, 1, 1'), 1.97
1
3
(
s, 6H), 1.76 (s, 6H); C NMR (100 MHz, DMSO-d ) δ 168.4 (2C, n, n'), 166.8 (2C,
6
9
1
, 9'), 150.7 (2C, 10, 10'), 150.3 (1C, p), 149.4 (2C, 13, 13'), 149.0 (2C, 14, 14'),
48.3 (2C, a, a'), 145.9 (1C, u), 143.6 (1C, t), 142.9 (1C, r), 140.9 (2C, 2, 2'), 139.2
(2C, 5, 5'), 138.6 (1C, x), 137.8 (1C, v), 137.2 (2C, c, c'), 135.0 (2C, d, g), 131.0 (2C,
e, f'), 130.8 (2C, 11, 11'), 120.0 (2C, 12, 12'), 129.4 (1C, j), 128.7 (21C, k), 128.0 (1C,
o), 127.9 (1C, i), 127.6 (1C, l), 127.0 (1C, b), 126.3 (1C,b'), 125.8 (1C, 1), 125.4 (1C,
1
'), 124.4 (1C, 6), 123.0 (1C, 6'), 121.3 (1C, h), 26.0 (2C, 7, 7'), 25.0 (2C, 8, 8');
+
HRMS (ESI) m/z: calcd for C H N SRu [M-2ClO -H] , 945.2070; found 945.2088.
5
5
39
8
4
1
0

ACCEPTED MANUSCRIPT
Caution: Perchlorate salts of metal compounds with organic ligands are potentially
explosive, and only small amounts of the material should be prepared and handled
with great care.
2
.3. Ruthenium(II) complexes-CT-DNA binding
The DNA-binding experiments were performed at room temperature. Buffer A [5
mM Tris-HCl, 50 mM NaCl, pH 7.0] was used for absorption titration, and viscosity
measurements. Buffer B (50 mM Tris-HCl, 18 mM NaCl, pH 7.2) was used for DNA
photocleavage experiments. Solutions of CT DNA in buffer A gave a ratio of UV-Vis
absorbance of 1.8~1.9:1 at 260 and 280 nm, indicating that the DNA was sufficiently
free of protein [40]. The concentration of DNA was determined
−1
−3
spectrophotometrically (Ɛ260 = 6600 M cm ) [41].
The absorption titrations of the complex in buffer were performed using a fixed
concentration (5.0 µM) for complex to which increments of the DNA stock solution
were added. The intrinsic binding constant K, based on the absorption titration, was
measured by monitoring the changes in absorption at the MLCT (metal-to-ligand
charge transfer) band with increasing concentration of DNA using the following
equation [42].
[
DNA]/(  ) [DNA]/(  ) 1/[K ((  )]
(1)
a
f
b
f
b
b
f
Where [DNA] is the concentration of DNA in base pairs,  a
,
 f and  b
correspond to the apparent absorption coefficient Aobsd/[Ru], the extinction coefficient
for the free ruthenium complex and the extinction coefficient for the ruthenium
11

ACCEPTED MANUSCRIPT
complex in the fully bound form, respectively. In plots of [DNA]/(ε -ε ) versus [DNA],
a
f
K is given by the ratio of slope to the intercept.
b
The emission spectra of the complexes in buffer were performed using a fixed
concentration (5 μM) for complexes to which increments of the CT DNA stock
solution were added. [Ru]-DNA solutions were allowed to incubate for 5 min before
the fluorescence emission spectra were recorded. The intrinsic binding constant (Kb)
of each Ru (II) complexes to CT DNA was obtained by the following equations:
2
2
1/2
(
F -F )/(F -F ) = (b-(b -2K C [DNA]/s) /2K C
(2)
(3)
a
f
b
f
b
t
b t
b = 1 + K C + K [DNA]/2s
b
t
b
where F , F , and F refer to the fluorescent intensities of the complex under a
a
f
b
given DNA concentration, in the absence of DNA and in the fully DNA-bound form
at 590 nm, respectively. C is the concentration of the Ru(II) complex. [DNA] is the
t
DNA concentration in nucleotides and s is the binding site size of complexes with
DNA in base pairs. Both the binding constant K and the binding site size (s) can be
b
obtained by nonlinear least-squares fit analysis using equations (2) and (3).
Viscosity measurements were carried out using an Ubbelodhe viscometer
maintained at a constant temperature at 25.0 ( 0.1) °C in a thermostatic bath. DNA
samples approximately 200 base pairs in average length were prepared by sonication
to minimize complexities arising from DNA flexibility [43]. Flow time was measured
with a digital stopwatch, and each sample was measured three times, and an average
flow time was calculated. Relative viscosities for DNA in the presence and absence of
0
0
complex were calculated from the relation η = (t – t )/t , where t is the observed flow
1
2

ACCEPTED MANUSCRIPT
0
time of the DNA-containing solution and t is the flow time of buffer alone [44,45].
1
/3
The change in the viscosity was presented as (/)
versus binding ratio
[
Ru]/[DNA] [46], where  is the viscosity of DNA solution in the presence of

complexes and  is the viscosity of DNA solution alone.


2
.4. Cytotoxicity assay in vitro
Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay
procedures were used [47,48]. Cells were placed in 96-well microassay culture plates
3
(
8 × 10 cells per well) and grown overnight at 37 °C in a 5% CO incubator. The
2
tested compounds were then added to the wells to achieve final concentrations
–
6
–4
ranging from 10 to 10 M. Control wells were prepared by addition of culture
medium (100 μL). The plates were incubated at 37 °C in a 5% CO incubator for 48 h.
2
–
1
Upon completion of the incubation, stock MTT dye solution (20 μL, 5 mg·mL ) was
added to each well. After 4 h, DMSO (100 μL) was added to solubilize the MTT
formazan. The optical density of each well was then measured with a microplate
spectrophotometer at a wavelength of 490 nm. The IC values were calculated by
5
0
plotting the percentage viability versus concentration on a logarithmic graph and
reading off the concentration at which 50% of cells remained viable relative to the
control. Each experiment was repeated at least three times to obtain the mean values.
2
.5. Apoptosis assessment by AO/EB staining
A549 cells were seeded onto chamber slides in six-well plates at a density of 2 ×
1
3

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5
1
0 cells per well and incubated for 24 h. The cells were cultured in RPMI 1640
o
containing 10% of FBS and incubated at 37 C in 5% CO . The medium was removed
2
and replaced with medium (final DMSO concentration, 0.05% v/v) containing the
complexes (4.5 µM) for 24 h. The medium was removed again, and the cells were
washed with ice-cold phosphate buffer saline (PBS), and fixed with formalin (4%,
w/v). Cell nuclei were counterstained with acridine orange (AO) and ethidium
-1
-1
bromide (EB) (AO: 100 µg mL , EB: 100 µgmL ) for 10 min. The cells were
observed and imaged with a fluorescence microscope (Nikon, Yokohama, Japan) with
excitation at 350 nm and emission at 460 nm.
2
.6. Reactive oxygen species (ROS) levels studies
The levels of the ROS in A549 cells induced by the complexes were measured
using the fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The
5
cells were seeded in a 12-well plate with 2 × 10 cells each well, and incubated
overnight. After being treated with different concentration of the complexes for 24 h,
the cells were washed twice with cold PBS and subsequently prestained with
o
DCFH-DA (10 mM) and incubated at 37 C in the dark for 30 min. Afterward, the
treated cells were washed two times with cold PBS, and stained with Hoechest 33342
o
for 20 min in the dark at 37 C. Finally, the cells were washed twice with PBS, and
then imaged and quantitatively analyzed using a confocal fluorescence microscope.
2
.7. The change of mitochondrial membrane potential assay
5
A549 cells were inoculated at a density of 2 × 10 cells/well in 12-well plates and
1
4

ACCEPTED MANUSCRIPT
o
incubated with or without different concentration of the complexes for 24 h at 37 C,
and then washed two times with PBS. Afterward, JC-1 dye (1 μg/mL) was added and
o
incubated for 20 min in the dark at 37 C. After being washed with PBS, the cells
were suspended in PBS, and observed under an ImageXpress Micro XLS system.
2
.8. Location assay of the complex in the mitochondria
4
A549 cells were placed in 12-well microassay culture plates (4 × 10 cells per
o
well) and grown overnight at 37 C in a 5% CO incubator. 1.0 µM of the complexes
2
o
were added to the wells at 37 C in a 5% CO incubator for 4 h and further
2
®
o
co-incubated with MitoTracker Deep Green FM (150 nM) at 37 C for 0.5 h. Upon
completion of the incubation, the wells were washed three times with ice-cold PBS.
After discarding the culture medium, the cells were imaged under a fluorescence
microscope.
2
.9. Matrigel invasion assay
BD Matrigel invasion chamber was used to investigate cell invasion according to
4
the manufacturer's instructions. A549 cells (4 × 10 ) in serum free medium
containing different concentrations of the complexes were seeded into the top
chamber of the two-chamber Matrigel system. RPMI 1640 medium (20% FBS) was
added into the lower chamber. The cells were allowed to invade for 24 h. After
incubation, non-invading cells were removed from the upper surface and cells on the
lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal
1
5

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violet. The membranes were photographed and the invading cells were counted under
a light microscope. The mean values from three independent assays were calculated.
3
. Results and discussion
3
.1. In vitro cytotoxicity assay
The MTT method was used to evaluate the antiproliferative activities of the Ru(II)
complexes against A549, HepG-2, SGC-7901 and Hela cancer cells, in comparison
with cisplatin as a positive control. The half maximal inhibitory concentration (IC )
5
0
values are summarized in Table 1. As expectation, the ligand shows low cytotoxic
activity (＞100 μM) against selected cancer cell lines. In addition, the complex
Ru(II)-4 displays excellent antitumor activity toward all selected cancer cell lines.
Complexes Ru(II)-1, Ru(II)-2 and Ru(II)-3 have poor cytotoxic activity against
HepG-2 cell lines. It is noteworthy that Ru(II) complexes exhibited well activity
against A549 cells with IC values of 9.3 ± 1.2 (Ru(II)-1), 12.1 ± 1.6 (Ru(II)-2),
5
0
1
0.3 ± 1.6 (Ru(II)-3) and 8.9 ± 1.2 (Ru(II)-4) μM, respectively, which are similar to
the anticancer activity of cisplatin. Thus, the complexes exhibits higher cytotoxicity
than ruthenium(II) complexes [Ru(ttbpy) (MHPIP)](ClO ) (IC = 33.8 ± 4.0 μM)
2
4 2
50
[
49] and [Ru(bpy) (HDPIP)](ClO ) (13.3 ± 1.1 μM) [50]. Therefore, we conclude
2 4 2
when BTPIP bonded ruthenium(II) to form complexes, the cytotoxic activity can be
greatly increased, and the Ru(II) complexes reveals high inhibitory effect on the cell
growth in A549, SGC-7901 and Hela cells and may be used as a potent
broad-spectrum anticancer drugs. Because all the Ru(II) complexes is sensitive to
1
6

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A549 cells, this cells were selected for the following experiments.
3
.2. Apoptosis assay with AO/EB and Annex V/PI double staining methods
Apoptosis induced by the Ru(II) complexes in A549 cells was investigated using
both qualitative and quantitative methods. The morphological changes of A549 cells
were obtained using AO/EB doubled staining methods [51]. As shown in Fig. 1, in the
control (a), the living cells show bright green with intact cell nuclei. After the
treatment of A549 cells with 4.5 μM of complexes Ru(II)-1, Ru(II)-2, Ru(II)-3 and
Ru(II)-4 for 24 h, the A549 cells with apoptotic features such as nuclear shrinkage,
cell blebbing and chromatin condensation were detected (Fig. 1 b, c, d, e), The results
revealed the Ru(II) complexes triggered apoptosis in A549 cells.
To further quantitatively analyze the extent of the four Ru(II) complexes on the
apoptosis, flow cytometry technique was employed to investigate the percentage in
the early and late apoptosis cells. As shown in Fig. 2, the total percentage of apoptotic
cells (sum of early and late apoptosis cells) in A549 cells increased from 2.37% (a) to
1
0.68% (b), 11.73% (c), 7.08% (d) and 20.99% (e) after treatment with complexes
Ru(II)-1, Ru(II)-2, Ru(II)-3 and Ru(II)-4, respectively. From these observations, it
can be deduced that the Ru(II) complexes induce obvious apoptosis in A549 cells and
the apoptosis effects follow the order Ru(II)-4 > Ru(II)-2 > Ru(II)-1 > Ru(II)-3.
3
.3. Intracellular reactive oxygen species levels determination
ROS can affect mitochondrial membrane potential and then cause a chain of
1
7

ACCEPTED MANUSCRIPT
mitochondria-associated events, such as apoptosis [52]. Therefore, it was of interest to
investigate whether the ruthenium(II) complexes would lead to increased ROS
generation. Herein, the ROS levels were investigated using a DCFH-DA
(2′,7′-dichlorodihydrofluorescein diacetate) probe. It is well known that DCFH-DA is
cleaved by intracellular esterase into its non-fluorescent form (DCFH), and DCFH is
oxidized by ROS to generate a fluorescent product DCF (dichlorofluorescein).
Consequently, the fluorescence intensity of DCF can reflect the content of
intracellular ROS [53]. A549 cells were treated with 4.5 μM of different Ru(II)
complexes for 24 h, stained with DCFH-DA and detected by ImageXpress Micro XLS
system. As described in Fig. 3, in the control (a), no obvious fluorescence was
observed. However, after A549 cells were treated with Rosup (positive control, b),
Ru(II)-1 (4.5 μM, c), Ru(II)-2 (4.5 μM, d), Ru(II)-3 (4.5 μM, e) and Ru(II)-4 (4.5
μM, f) for 24 h, the obvious green fluorescence were found, which indicates that the
Ru(II) complexes can increase the contents of ROS. To quantitatively investigate the
effect of concentration of the Ru(II) complexes on the ROS levels, The DCF
fluorescent intensity was tested and depicted in Fig. 4. In the control, the fluorescent
intensity of DCF is 1.9. However, A549 cells were treated with 9.0 μM of complexes
Ru(II)-1, Ru(II)-2, Ru(II)-3 and Ru(II)-4, the fluorescent intensities are 35.4, 12.7,
2
0.0 and 57.6, respectively. Compared the effect with the control, the fluorescent
intensities of DCF grow 18.6, 6.7, 10.5, and 30.3 times than the original. Furthermore,
the content of ROS shows a dose-dependent manner.
·‒
ROS include superoxide anion (O ), hydrogen peroxide (H O ) and nitric oxide
2
2
2
1
8

ACCEPTED MANUSCRIPT
(
NO), etc [54]. Among them, superoxide anion plays a vital role in cancer cell death
55]. Subsequently, the level of superoxide anion was investigated by DHE assay. The
[
non-fluorescent DHE could enter into cells freely and interact with superoxide anion
to form the membrane-impermeant ethidium cation, which becomes fluorescent upon
intercalating DNA, thus the fluorescence intensity of ethidium-DNA can indicate the
•–
level of intracellular O [56] As described in Fig. 5, Comparing with the control (a),
2
the treatment of A549 cells with complexes Ru(II)-1 (b), Ru(II)-2 (c), Ru(II)-3 (d)
and Ru(II)-4 (e) led to an obvious increase in the red fluorescent points. In addition,
the red fluorescence intensity was determined by ImageXpress Micro XLS system
and is depicted in Fig. 6, all the ruthenium(II) complexes can enhance the levels of
•–
O2 in a dose-dependent manner.
The content of nitric oxide is closely related to cancer cells apoptosis and necrosis
[
57]. 3-Amino, 4-aminomethyl-2',7'-difluorescein, diacetate (DAF-FM DA) was used
as a fluorescent indicator of intracellular nitric oxide (NO). As described in Fig. 7,
treatment of A549 cells with 4.5 μM of Ru(II)-1 (b), Ru(II)-2 (c), Ru(II)-3 (d), and
Ru(II)-4 (e) for 24 h, more bright green fluorescent points were detected compared
with the control (a), which confirms that the levels of NO was increased. The
fluorescence intensity of DAF-FMDA was tested using ImageXpress Micro XLS
system. As described in Fig. 8, exposure of A549 cells to 4.5 and 9.0 μM of Ru(II)
complexes led to an remarkable increase in green fluorescence intensity of 4.96 and
1
0.04 times for Ru(II)-1, 3.43 and 7.53 times for Ru(II)-2, 4.18 and 9.03 times for
Ru(II)-3, 4.28 and 11.76 times for Ru(II)-4 than that of control, respectively. This
1
9

ACCEPTED MANUSCRIPT
further confirms that the Ru(II) complexes can increase the content of nitric oxide.
Furthermore, the level of NO shows a dose-dependent manner.
3
.4. Location assay of the complexes and mitochondrial membrane potential (MMP)
analysis
Mitochondria play a vital role in a series of cellular processes, deciding the fate of
a cell by controlling the process of apoptosis [58]. In order to research the location of
®
the ruthenium(II) polypyridyl complexes in the mitochondrial, Mito Tracker Deep
Green FM (ThermoFisher, 100 nM) was used as a green fluorescent dye. As shown in
Fig. 9, in the control, the mitochondria were stained in bright green. The treatment of
A549 cells with 1.0 μM of Ru(II)-1, Ru(II)-2, Ru(II)-3 and Ru(II)-4 for 4 h, the
Ru(II) complexes emits red fluorescence. The merge of the green and red
fluorescence reveals that the Ru(II) complexes could reach the cytoplasm through the
cell membrane and accumulate in the mitochondria. To further explore the possible
anticancer mechanism, we studied the effects of Ru(II) complexes on the
mitochondrial membrane potential (MMP) of A549 cells using JC-1
(5′,6′,6′-tetrachlore-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine
iodide).
The
accumulation of JC-1 in mitochondria is potential-dependent, and it is demonstrated
by a shift in its fluorescence emission from red (~590 nm) to green (~525 nm) [59].
Image of A549 cells obtained using fluorescence microscope. As depicted in Fig. 10,
in the control (a), JC-1 emits red fluorescence corresponding to high MMP. After
A549 cells were exposed to CCCP (carbonylcyanide-m-chlorophenylhydrazone, b,
2
0

ACCEPTED MANUSCRIPT
positive control), Ru(II)-1 (4.5 μM, c), Ru(II)-2 (4.5 μM, d), Ru(II)-3 (4.5 μM, e)
and Ru(II)-4 (4.5 μM, f) for 24 h, JC-1 emits green fluorescence corresponding to
low MMP. These findings suggest that treatment with Ru(II) complexes causes a
remarkable decrease of MMP which is evidenced by an opposite fluorescence
emission shift from red to green [60]. Furthermore, the ratio of red/green fluorescent
intensity was also detected by ImageXpress Micro XLS system. As shown in Fig. 11,
in the control and in the presence of CCCP, the ratios of red/green are 0.94 and 0.49,
respectively. Treatment of A549 cells with different concentrations of Ru(II)
complexes led to an decrease of the ratio of red/green. It is further proved that the
Ru(II) complexes can induce the decrease of membrane potential of mitochondria.
3
.5. Transwell cell migration and invasion assay
The migration and invasion are the vital factors in the process of cancer metastasis
[
61-63]. According to the observations above, designed Ru(II) complexes can induce
apoptosis and effectively inhibit the A549 cells growth. Therefore, it is reasonable to
deduce that Ru(II) complexes may have the capability to inhibit the migration of
A549 cells. We carried out Boyden chamber invasion assay to confirm the effect of
Ru(II) complexes on the invasive ability of A549 cells. As seen in Fig. 12, invasive
ability of A549 cells was reduced by the treatment with 4.5 μM of different Ru(II)
complexes (b, c, d, e) with the control (a). Then, the percentage of Ru(II) complexes
inhibiting the cell invasion is calculated in Fig. 13. A549 cells were treated with 9.0
μM of complexes Ru(II)-1, Ru(II)-2, Ru(II)-3 and Ru(II)-4 for 24 h, and the
2
1

ACCEPTED MANUSCRIPT
percentage of inhibiting the cell invasion reaches 63.4%, 51.9%, 54.1% and 65.7%,
respectively. Besides, it is clear that the Ru(II) complexes inhibited cell invasion with
a dose-dependent manner.
3
.6. Cell cycle arrest studies
The inhibition of cell viability by anticancer drugs can result from cycle cell arrest,
induction of apoptosis, or a combined action of these two mechanisms [64]. We
therefore investigated whether target Ru(II) complexes altered the cell cycle of A549
cells and induced apoptosis of A549 cells using flow cytometric analyses. As seen in
Fig. 14, in the control (a), the percentage in the cell cycle at S phase is 24.78%.
Treatment of A549 cells with different Ru(II) complexes, the percentages in the at S
phase are 38.83% (b), 41.24% (c), 34.58% (d) and 40.73% (e), respectively, An
obvious increase of 14.05% for Ru(II)-1, 16.46% for Ru(II)-2, 9.80% for Ru(II)-3
and 15.95% for Ru(II)-4 in the percentage at S phase was detected, at the same time,
a reduction of 10.82% for Ru(II)-1, 15.54% for Ru(II)-2, 8.39% for Ru(II)-3 and
1
4.31% for Ru(II)-4 in the cell at G /G phase compared with the control were
0 1
discovered. These results demonstrate that the antiproliferative mechanism induced by
Ru(II) complexes on A549 cells was a S phase arrest. In addition, a population of
sub-G -phase cells (Apo), the characteristic of apoptosis, was increased dramatically
1
with the incubation of Ru(II) complexes.
3
.7. Complexes-CT DNA binding studies
2
2

ACCEPTED MANUSCRIPT
Electronic absorption spectroscopy is the effective way to investigate the binding
mode of complexes with DNA by testing changes in absorption intensity and position
of the bands [65]. The electronic absorption spectra of complexes Ru(II)-1, Ru(II)-2,
Ru(II)-3 and Ru(II)-4 mainly consist one or two resolved bands in the range of
3
00-600 nm. Fig. S1 shows spectra obtained at increasing amounts of CT DNA (0.75
μM) to Ru(II)-1 (5.0 μM), Ru(II)-2 (5.0 μM), Ru(II)-3 (5.0 μM) and Ru(II)-4 (5.0
μM) (10 μL of CT DNA was added to corresponding solutions each time, see the
Supporting Information for details). As the CT DNA concentration increased, the
MLCT (metal-to-ligand charge transfer) bonds of Ru(II)-1 at 467 nm, Ru(II)-2 at 465
nm, Ru(II)-3 at 476 nm and Ru(II)-4 at 479 nm exhibit hypochromism of about
2
6.4%, 26.6%, 16.2%, 21.0%, and bathochromism of 5, 6, 2 and 2 nm, respectively.
6
-1
The K values of Ru(II)-1, Ru(II)-2, Ru(II)-3 and Ru(II)-4 are 1.2 ×10 M , 3.81 ×
b
6
-1
6
-1
5
-1
1
0 M , 1.09 × 10 M and 5.41 × 10 M , respectively, which are less than that of
2
+
6
-1
2+
the complexes [Ru(phen) (dppz)] (5.1 × 10 M ) [66] and [Ru(dmb) (dppz)] (4.5
2
2
6
-1
×
×
10 M ) [67], but were higher than that of complexes [Ru(phen) bppp](ClO ) (5.51
2 4 2
5
-1
2+
6
10 M ) [68] and [Ru(bpy) (ppd)] , (1.3 × 10 ) [69].
2
Fluorescence emission spectra are the most common and most effective way to
analyze complex-nucleic acid interactions [70]. The emission intensities of the
Ru(II)-1, Ru(II)-2, Ru(II)-3 and Ru(II)-4 from their MLCT excited states upon
excitation at 594, 577 and 604 nm were found to depend on CT DNA concentration.
As shown in Fig. S2, the emission intensities of Ru(II) complexes grow to 1.9
(Ru(II)-1), 2.4 (Ru(II)-2) and 1.4 (Ru(II)-3) times larger than the original,
2
3

ACCEPTED MANUSCRIPT
respectively. The results indicates that target Ru(II) complexes can effectively interact
with CT DNA, and the Ru(II) complexes can be protected efficiently by the
hydrophobic environment inside the CT DNA helix [71]. The intrinsic binding
constants K of Ru(II)-1, Ru(II)-2 and Ru(II)-3 to CT DNA calculated from
b
5
-1
5
-1
5
equations (eq 2) and equations (eq 3) are 7.68 × 10 M , 8.91 × 10 M , 5.56 × 10
-1
2+
M respectively, which are higher than that of the complexes Δ-[Ru(bpy) PBIP]
2
5
-1
2+
5
-1
(
4.1 × 10 M ) and Λ-[Ru(bpy) PBIP] (2.6 × 10 M ) [72].
2
To further confirm the mode of designed Ru(II) complexes with CT DNA,
viscosity measurements of CT DNA solutions were carried out in the presence and
absence of these complexes [13]. Viscosity of CT DNA is sensitive to its length.
Compounds are expected to elongate the double helix to accommodate the ligands in
between the base leading to an increase in the viscosity of CT DNA [73]. The effects
of target Ru(II) complexes Ru(II)-1, Ru(II)-2, Ru(II)-3 and Ru(II)-4 on the viscosity
of CT DNA were seen in Fig. 15. On increasing the amount of Ru(II) complexes, the
relative viscosity of CT DNA solution increases steadily. The increased degree of
viscosity, which may depend on its affinity to CT DNA follows order of Ru(II)-2 >
Ru(II)-1 > Ru(II)-3 > Ru(II)-4. These results indicated that the designed Ru(II)
complexes bind CT DNA through a classical intercalation model, which was
consistent with binding constants.
4
. Conclusions
A novel ligand BTPIP and its four Ru(II) polypyridyl complexes
2
4

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[
[
Ru(bpy) (BTPIP)](ClO )
(Ru(II)-1), [Ru(phen) (BTPIP)](ClO )
(Ru(II)-2),
2
4 2
2
4 2
Ru(dmb) (BTPIP)](ClO ) (Ru(II)-3) and [Ru(dmp) (BTPIP)](ClO ) (Ru(II)-4)
2
4 2
2
4 2
were synthesized and characterized. Designed Ru(II) polypyridyl complexes interact
with CT DNA most probably by intercalative binding mode. They exhibited good
activity against selected cancer cells, especially A549 cells. In addition, AO/EB and
Annex V/PI staining assay demonstrates that the complexes can obviously induce the
apoptosis of A549 cells. Detailed studies on the Ru(II) complexes, suggest that they
localizes mainly in the mitochondria, causing a decrease in mitochondrial membrane
potential and ROS release into cytoplasm, which further induced mitochondria
dysfunction and the onset of apoptotic cell death mechanism. Further anticancer
mechanistic studies reveal that these complexes can significantly inhibit cell invasion
and induce cell cycle arrest of A549 cells at S phase. According to the results of these
experiments, we suggest that designed Ru(II) complexes may be a candidate for
further evaluation as a chemopreventive and chemotherapeutic agent for human lung
cancer.
Acknowledgements
The authors thank the Guangxi Natural Science Foundation (2018GXNSFBA050024),
the Ph. D. Scientific Research Foundation of Guilin University of Technology, and
Key Laboratory of Electrochemical and Magneto-chemical Function Materials.
2
5

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